U.S. patent number 4,487,715 [Application Number 06/396,578] was granted by the patent office on 1984-12-11 for method of conjugating oligopeptides.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Pradip K. Bhatnagar, Danute E. Nitecki.
United States Patent |
4,487,715 |
Nitecki , et al. |
December 11, 1984 |
Method of conjugating oligopeptides
Abstract
Novel methods are provided for preparing peptide compounds
involving functionalizing an available amino group with a carboxy
group, esterifying the carboxy group with an hydroxylic compound
which forms an ester which is stable under the conditions of
cleavage of the oligopeptide from said resin, but which ester is
capable of forming a peptide bond with an amino group in an aqueous
medium, and without separation, either allowing the oligopeptide to
react with itself or with a polypeptide compound.
Inventors: |
Nitecki; Danute E. (San
Francisco, CA), Bhatnagar; Pradip K. (Santa Clara, CA) |
Assignee: |
The Regents of the University of
California (Berkeley, CA)
|
Family
ID: |
23567816 |
Appl.
No.: |
06/396,578 |
Filed: |
July 9, 1982 |
Current U.S.
Class: |
530/334;
525/54.11; 530/342 |
Current CPC
Class: |
C07K
1/08 (20130101); C07K 1/006 (20130101) |
Current International
Class: |
C07K
1/00 (20060101); C07K 1/08 (20060101); C07C
103/52 (); C08L 089/00 () |
Field of
Search: |
;260/112.5R
;525/54.11 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Phillips; Delbert R.
Attorney, Agent or Firm: Rowland; Bertram I.
Claims
What is claimed is:
1. An improved method for preparing a polypeptide product capable
of forming a peptide bond in an aqueous medium, said method being
of the type wherein an oligopeptide is formed by the sequential
addition of amino acids to an initial amino acid which is bound to
a 4-alkoxybenzyl alcohol resin or chloromethyl resin through an
ester linkage, said improvement comprising:
reacting a cyclic carboxylic acid anhydride with an available amino
group on the oligopeptide to form an amic acid to provide a single
free reactive carboxyl group on said oligopeptide;
esterifying said carboxyl group with an hydroxyl compound selected
from the group consisting of 4-hydroxy-3-nitrobenzenesulfonic acid
salt, pentachlorophenol, N-hydroxy succinimide, and p-nitrophenol
to provide an activated ester stable to cleavage of the
oligopeptide from the resin and capable of forming a peptide bond
in an aqueous medium;
cleaving said oligopeptide from said resin employing hydrofluoric
acid or trifluoroacetic acid in an inert organic solvent to provide
a mixture of said resin and said oligopeptide in an acid
solution;
neutralizing said acid solution to a mildly basic pH in the absence
or presence of a peptide having an available amino group, whereby
said oligopeptide reacts either (1) intramolecularly or
intermolecularly with itself or (2) with said amino containing
peptide to form a peptide bond.
2. A method according to claim 1, where said anhydride is succinic
anhydride.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
There is an ever increasing need to covalently attach small
molecules (haptens, enzyme substrates or inhibitors, reporter
groups, etc.) to larger molecules (carriers, enzymes, insoluble
matrices, etc.) for a variety of applications (vaccines, antibody
production, immunoassays, isolation and separation techniques,
etc). In many instances the small molecule is a peptide, which can
be synthesized by current methodology. However, the polyfunctional
nature of both the peptides and the larger molecule (e.g.,
proteins) does not allow for precisely controlled covalent
attachment. The most commonly used coupling reagents are for the
most part bifunctional, with the two functions of equal activity,
frequently being the same functionality. Conventional reagents
include dialdehydes, carbodiimides, diimidates and diesters, which
tend to cross-link the proteins intramolecularly and
intermolecularly, resulting in a low efficiency of the desired
peptide carrier linkage, as well as substantial modification of the
carrier molecules.
Since activation in aqueous media is very nonproductive, the
stoichiometry of the conjugation is usually quite poor, i.e., only
a small portion of the peptide used becomes attached to the larger
molecule. Moreover, it is frequently desirable to know which
functional group in the small molecule was utilized for linkage to
the larger molecule.
Thus, there is an important need to develop methods which allow a
well designed coupling site for the smaller molecule and are
stoichiometrically acceptable for the conservation of materials in
limited supply.
2. Description of the Prior Art
U.S. Pat. No. 4,127,526 describes the preparation of oligopeptides
on chloromethylated resins. 4-Hydroxy-3-nitrobenzenesulfonic acid
salt esters are reported in Klausner et al., in Peptides
Proceedings of the Fifth American Peptide Symposium, Goodman, M.
and Meienhofer, J., eds. John Wiley & Sons, New York, pp.
536-538. The N-9-fluorenylmethyloxycarbonyl (FMOC) is described by
Chang et al., Int. J. Peptide and Protein Res. (1980)
15:485-494.
SUMMARY OF THE INVENTION
Method and compositions are provided for preparing activated
oligopeptides. A peptide chain is elongated while bound to a solid
support, followed by functionalization to form an active ester
group which is stable to cleavage from the support. The activated
oligopeptide is cleaved from the support and may be used in aqueous
media for conjugation to amino group containing polyfunctional
molecules, for polymerization or cyclization.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In accordance with the subject invention, activated oligopeptides
are prepared for conjugation to functionalities capable of forming
a stable bond with an active ester group. An amino containing
molecule is bound to a support and an oligopeptide chain formed by
the successive addition of amino acids. The individual amino acids
are appropriately protected and blocking groups removed in
accordance with conventional techniques. The resulting
oligopeptide, either has an available carboxyl group or a carboxyl
group is introduced by bonding an appropriate compound having a
carboxyl group with an available functionality, e.g. amino,
particularly the terminal amino. The carboxyl group is then further
functionalized to provide an active ester which is capable of
forming a covalent bond in an aqueous medium. The activated
oligopeptide is then cleaved from the column and may be used
directly without seperation for covalent bonding to another
compound, particularly one having an amino functionality, such as a
polypeptide or protein.
In performing the subject method, the reagents which are involved
are: (1) the solid support; (2) the amino acids and their
appropriate protecting groups, as well as the means for
deprotection; (3) the active ester functionality formed from an
available carboxy group on the oligopeptide, and as appropriate, a
reagent for introducing the carboxy group; (4) cleavage reagents
for removing the oligopeptide from the solid support; and (5)
reagents for conjugating the activated polypeptide to another
molecule. In discussing the subject invention, the various reagents
will be considered individually followed by a detailed description
of the method.
MATERIALS
Solid Support
The solid support may take any convenient form, such as particles,
filters, wall surfaces, or the like. The significant factors for
the solid support are its inertness, except for the functionality
involved with linking the oligopeptide; ease and manner of removal
of the oligopeptide; and absence of non-specific binding. A number
of different polymers have found use, particularly latexes, such as
polystyrene and polyacrylamides; polyethylene glycol resins, etc.
The polymers may be cross-linked to various degrees, usually
lightly cross-linked. Alternatively, inorganic materials may be
used, such as Bioglas, silicones, etc.
A variety of groups have been employed to provide for cleavable
anchor linkages of peptide to the solid support. Included among
these groups are chloromethyl, 4-alkoxybenzyl alcohol,
benzhydrylamine and the like. Many of the polymers, particularly
the modified styrene latexes, are commercially available. Varying
numbers of linkage groups per polymer unit are available, but are
not critical to this invention.
The amino acids which are employed may be naturally occurring or
synthetic. For the most part, the amino acids will be alpha-amino
acids, but the amino group need not be at the alpha position and
may be at any position. The amino acid may be aliphatic, alicyclic,
aromatic or heterocyclic. The amino acids may be of the D- or L-
configuration or a racemic mixture.
A wide variety of protective groups may be conjugated to the amino
group to provide for the stepwise addition of the amino acids.
Illustrative protective groups for protecting amino groups include
benzyloxycarbonyl (Z), tert-butyloxycarbonyl (BOC),
fluorenylmethyloxycarbonyl (FMOC), isobutyryloxycarbonyl,
adamantyloxycarbonyl, o-nitrophenylthiocarbonyl, chloro- or
nitrobenzyloxycarbonyl, or the like. The hydroxyl-protecting groups
will be for the most part benzyl or tert.-butyl. For carboxyl
groups, benzyl or substituted benzyl, e.g., chloro, bromo, or
nitro, or tert.-butyl may be employed. The oxy protecting group
will provide ethers and esters. In some instances, it may be
feasible to employ protective groups, such as sugars, which may be
cleaved enzymatically.
The binding of the amino acids to the support, the elongation of
the peptide chain, and the removal of the protective groups will
follow conventional procedures. Removal of the protective groups
may include hydrogenolysis, strong acid catalyzed removal, e.g.,
trifluoracetic acid, hydrochloric acid, hydrobromic acid,
methanesulfonic acid, etc., in an organic non-protic or protic
solvent, or other technique, e.g. enzyme catalysed removal.
In some situations, it will be feasible to employ different
protective groups, particularly between the alphaamino group and a
side chain substitutent, where the protective groups may be
selectively removed. In this manner, the activated moiety can be
conjugated to other than the terminal amino group and at a specific
site.
Condensation of the carboxyl group with the deprotected amino group
to elongate the oligopeptide will follow conventional conditions,
normally employing an activated carboxyl group. The activation may
be by use of an activated ester (these esters will be described
below), carbodiimide, mixed anhydride, or the like.
The manner of providing the active ester for conjugation of the
oligopeptide to another molecule will vary depending upon whether
the oligopeptide has an available carboxy group, or such carboxy
group must be introduced. Available carboxy groups may be present
as a result of including in the oligopeptide aspartic acid,
glutamic acid, or other dicarboxylic amino acid which is not
conventionally found in naturally occurring polypeptides or
proteins. Where there is no carboxyl group, a carboxyl group will
have to be introduced. The introduction of a carboxyl group can
take many forms, depending upon the manner of linking the carboxy
group to the oligopeptide. The conventional functionalities which
are available for introduction of the carboxyl functionality will
include amino, mercapto and hydroxyl.
By appropriate employment of conventional protecting groups, one
can selectively protect a particular functionality to provide for a
unique site at which to introduce the carboxy functionality. The
carboxy functionality to be subsequently activated will be joined
by a linking group to the heteroatom nitrogen, oxygen, or
sulphur.
The linking group between the carboxy and the heteroatom will have
at least one carbon atom, usually not more than 20 carbon atoms,
more usually not more than 10 carbon atoms, generally from about 1
to 6 carbon atoms, and from 0 to 4, usually 0 to 2 heteroatoms,
which will be oxygen (as oxy, that is hydroxy or ether), nitrogen
(amino or amido, free of reactive hydrogens) and sulfur (thiol or
thioether). The linking group may be aliphatically saturated or
unsaturated, usually having not more than one site of aliphatic
unsaturation, e.g., ethylenic. The linking group may be aliphatic,
aromatic, alicyclic or heterocyclic.
Where an amino group is the configuration site, the
functionalization of the peptide chain with an activated ester is
readily and preferably achieved by conjugation of a cyclic
anhydride, which cyclic anhydride may be aliphatic, alicyclic,
aromatic or heterocyclic. In most instances, the aliphatic
anhydrides will find use, although in special situations
1,2-dicarboxylic acids bonded to a ring may find use. For the most
part, the dibasic carboxylic acids will have from 4 to 20 carbon
atoms, more usually from 4 to 12, and preferably from about 4 to 8
carbon atoms. The dicarboxylic acids may be substituted with a
variety of functional groups, such as oxy (hydroxy or ether),
cyano, halo, nitro, amino, (including alkyl substituted) amido, or
the like.
Illustrative anhydrides include succinic anhydride, glutaric
anhydride, phthalic anhydride, 1,2-cyclohexanedicarboxylic acid
anhydride, 4-nitrophthalic anhydride,
2-2,dimethyl-4,5-dicarboxy-1,3-dioxacyclopentane, maleic anhydride,
N-methyl 2,3-dicarboxyindole anhydride, etc.
Besides cyclic anhydrides mono-activated dicarboxylic acids may be
employed where the dicarboxylic acid is esterfied stepwise: once
before conjugation to the oligopeptide and then after conjugation
to the oligopeptide.
Rather than carboxyl groups, other acyl groups may be used such as
sulfonyl halides, phosphonyl halides, etc., where such acyl group
would offer a synthetic advantage or impart a desirable property to
the oligopeptide.
With acyl groups, the basic character of the amino group is lost.
In the case of the N-terminal amino group, this may be desirable.
However, for an amino group or an internal amino acid it may be
desirable to retain the basic character of the amino group. Various
procedures known in the literature may be used for linking a
carboxy group to an amino group. Activated halo groups may be
employed, particularly iodo and bromo, as in iodoacetic acid and
.alpha.-bromopropionic acid. Reductive amination may be employed
where an imine is formed with an aldehyde and the imine then
reduced with a metal hydride, e.g., cyanoborohydride. Illustrative
compounds include carboxyacetaldehyde, glucuronic acid,
galacturonic acid, 5-carboxypentanal, 3-carboxyacrylaldehyde, and
carboxymethoxyacetaldehyde
The carboxylic acid (including anhydride) compounds that are
employed should be free of groups which interfere with the
conjugation of the carboxylic acid compounds to the oligopeptide
and the formation of the activated ester. This may be as a result
of the absence of such groups or the presence of protective groups
which may be removed in conjunction with cleavage from the support
or be removed in a separate step from such cleavage.
Where mercaptide groups are involved, one may use active halides,
olefins such as are present in acrylic acid, or reagents having a
carboxy group and capable of forming a disulphide with a mercaptide
group.
With hydroxyl groups, ethers will be formed, normally with active
halides or active esters.
After providing for the carboxylic acid group, the free carboxyl
group is activated to form an active ester. With the cyclic
anhydride, the half amide acid (amic acid) is formed. By active
ester is intended an ester which is capable of forming a covalent,
usually a peptide, bond in an aqueous medium. Usually the bond will
be to a functionality normally present in a polypeptide. Various
hydroxyl componds have been employed to form these esters. These
hydroxyl componds are illustrated by N-hydroxy succinimide,
pnitrophenol, pentachlorophenol and
4-hydroxy-3-nitrobenzenesulfonic acid sodium salt.
Cleavage from the support may be achieved in a variety of ways,
depending upon the particular support. Conveniently, hydrogen
fluoride or trifluoroacetic acid may be employed or hydrogenolysis,
where the active ester group is retained and the oligopeptide
separated from the support.
METHOD
In performing the subject method, the first step is to conjugate
the initial amino acid to the support. The particular linkage will
be capable of surviving the procedures employed in elongating the
peptide chain. For the most part esters will be formed, the
conditions varying with the reactive functionality present on the
support. Conventional conditions are employed in accordance with
those suggested by the commercial supplier of the resin.
The peptides are prepared using standard solid-phase techniques.
The synthesis is commenced from the C-terminal end of the peptide,
using an alpha-amino protected amino acid. The resin can be
prepared by attaching the required alpha-amino acid to a
chloromethylated resin or an hydroxymethyl resin or benzhydrylamine
resin, an exemplary chloromethylated resin is sold under the trade
name BIO-BEADS XF-1 by Bio-Rad Laboratories, Richmond, California.
The preparation of an exemplary hydroxymethyl resin is described by
Bodonszky et al., Chem.Ind. (London) 38, 1597 (1966).
The alpha-amino protected amino acid can be coupled to the
chloromethylated resin according to the method described by Gisin,
Helv. Chim. Acta 56, 1476 (1973). Conveniently, cesium bicarbonate
may be employed. The alpha-amino protecting group may then be
removed by any conventional technique, such as trifluoroacetic acid
or hydrogen chloride in an organic solvent at room temperature.
After removal of the alpha-amino protecting group, the remaining
protected amino acids are added stepwise in the desired order. Each
protected amino acid is generally reacted in a three-fold excess
using an appropriate carboxyl group activator, such as an organic
solvent soluble carbodiimide e.g. dicyclohexyl diimide in a mixed
solvent, e.g., methylene dichloride-dimethyl formamide.
For further description of synthetic methods, including methods of
deprotection of amino groups see Barany and Merrifield, Solid-Phase
Peptide Synthesis "The Peptides, Analysis, Synthesis, Biology,"
Special Methods in Peptide Synthesis, Part A, Vol. 2, Gross and
Meienhofer, Eds., Academic Press, New York, 1980, pages 1-284;
Chang et al., Int. J. Peptide Protein Research (1980) 15: 485-494;
Meienhofer et al., Ibid (1979) 13:35-42.
After completion of the synthesis of the desired oligopeptide, the
oligopeptide may be partially or completely deprotected while
retaining the linkage to the resin. Where a carboxy group is
present in an oligopeptide, it will normally be present in
protected form and may be deprotected to provide the desired
carboxyl functionality. Where the carboxyl group to be activated is
present on the oligopeptide it will be deprotected. Where a
carboxyl group is not protected, one other functionality will be
deprotected for linking of the carboxyl functionality. There will
be one functionality which will be preferentially conjugated to the
carboxy group. By using different protective groups, one can
preferentially remove one protective group as distinguished from
another. Protective groups present on a carboxylic acid present in
the oligopeptide may or may not be retained, depending upon the
activity of the particular carboxylic acid. Where the carboxylic
acid is relatively unreactive, it may be deprotected, but where it
will compete with the carboxylic acid which is introduced, the
protective group will not only be retained, until at least the
active ester is formed, but potentially after reaction of the
active ester to form the final product.
Deprotection may involve acids of varying degrees of acidity, based
upon the particular acid and the solvent system, bases, of various
degrees of basicity, which may include hydroxylic bases, tertiary
amines, and the like, where aqueous or non-aqueous systems are
employed. Protective groups for carboxyl may include nitrobenzyl,
benzyl, tert.-butyl, or other esters. Besides hydrolytic
techniques, hydrogenolysis may also be employed.
Where a carboxyl group is to be introduced, the partially or
completely deprotected oligopeptide bound to the resin may now be
employed for functionalization. The particular conditions for
functionalization will vary with the compound used and the
functionality which reacts with the group on the oligopeptide with
an amino group when employing cyclic anhydrides, at least one
equivalent per equivalent of oligopeptide will be used, usually at
least 2, and amounts of 10 equivalents or more may be used,
although greater excesses are usually not desirable. An inert
organic solvent is normally employed, conveniently a
halo-hydrocarbon. The reaction is carried out under mild
conditions, generally ambient temperatures, desirably in the
presence of a catalyst e.g., 1-hydroxybenzotriazole. After
sufficient time for the reaction to occur, the resin may be
isolated and purified or may be used directly.
For reductive amination, the aldehyde-acid may be added to the
resin in a polar organic solvent, e.g., alkanol, at a pH
approaching neutrality or neutrality and after sufficient time for
formation of the imine, the solution is made mildly basic, about pH
7.2 to 7.5 and cyanoborohydride added in mild excess. Reduced
temperatures are normally employed and the reaction allowed to
proceed to completion.
In individual situations, haloalkylesters may be employed in the
presence of a polar organic solvent, particularly in the presence
of a tertiary amine.
After completion of the introduction of the carboxyl group, the
resin may now be washed, conveniently by repetitive washes with
inert solvents to remove the excess of the other reactants and any
materials which are nonspecifically adhered to the resin. The
washed resin may then be combined with an appropriate hydroxylic
compound for preparation of the ester. The ester is conventionally
prepared employing a polar carbodiimide in an inert organic
solvent, e.g., DMF and/or CH.sub.2 Cl.sub.2, where the activating
carbodiimide and hydroxylic compound are used in excess, usually at
least two-fold excess and usually less than about 5-fold excess.
Ambient conditions may be employed and the reaction allowed to
proceed until completion. The resulting products may then be
further purified, conveniently by washing with inert solvents, and
the product dried.
Significant to the subject invention is the manner in which the
oligopeptide is cleaved from the resin. The conditions employed
must distinguish between the ester linkage to the resin and the
activated ester to be employed for further reaction. Particularly,
a strong acid is employed at mild temperatures, generally from
about -20.degree. to 30.degree. C., more usually from 0.degree. to
about 25.degree. C., in an inert organic solvent, particularly an
aromatic solvent, more particularly an aromatic ether of from about
7 to 10 carbon atoms. Of particular interest as acids are those
which cannot react to form a covalent product with the various
functionalities present in the oligopeptide. These acids are
particularly exemplified by hydrofluoric acid and trifluoroacetic
acid. Relatively small amounts of the acid may be added, since the
acid serves a catalytic role. After the addition of the acid, the
reaction may then proceed for sufficient time for the oligopeptide
to be cleaved from the resin.
The resulting slurry may then be used directly for reaction of the
oligopeptide. The slurry is combined with the appropriate other
reactant or may react with itself, intramolecularly or
intermolecularly in an aqueous medium, generally at a mildly basic
pH, e.g. 8-10. The acid is conveniently neutralized with a mild
base e.g. borate carbonate, phosphate, etc. The product may then be
isolated and purified in accordance with conventional techniques,
e.g., chromatography, electrophoresis, etc. The oligopeptides may
be used for a wide variety of purposes. Internal cyclization can
lead to products having physiological activity, tyrocidine,
gramicidin S, and the like. The oligopeptide may be polymerized to
provide a repeating unit, which may be used in affinity columns, as
immunogens, and the like. Alternatively, the oligopeptide may be
used as a hapten and conjugated to an immunogen to produce
antibodies recognizing a specific determinant site of a polypeptide
or protein of interest. Various common immunogens include bovine
serum albumin, bovine gamma-globulin, keyhole limpet hemocyanin, or
the like.
The following examples are offered by way of illustration and not
by way of limitation.
EXPERIMENTAL
The following abbreviations will be used having the indicated
definitions: Boc, tert.-butyloxycarbonyl; DNP, 2,4-dinitrophenyl;
FMOC, N-9-fluorenylmethoxycarbonyl; BSA, bovine serum albumin; OSu,
N-hydroxy succinimide; ONp, p-nitrophenyl; PCP, pentachlorophenyl;
HNSA, 4-hydroxy-3-nitrobenzene sulfonic acid sodium salt; (P),
polymeric resin.
The peptide, Boc-Lys-(.epsilon.-DNP)-Ala-(P)((P) in this example is
chloromethyl resin) was synthesized and amino group deprotected and
neutralized by standard methods (Barany and Merrifield) supra. The
resin (5 g 0.32 mm Cl/g of peptide as determined by Ala
substitution; 1.6 meq total) was dispended in CH.sub.2 Cl.sub.2 and
treated with 8 meq of succinic anhydride (800 mg) and a catalyst,
1-hydroxybenzotriazole hydrate (1.08 g, 8 meq) with mixing. After
three hours, the polymer was washed 5.times.CH.sub.2 Cl.sub.2 ;
5.times.DMF; 5.times.CH.sub.2 Cl.sub.2 (about 35 ml each). A 100 mg
aliquot of the polymer was taken and analyzed by high voltage
electrophoresis and shown to have the expected properties. The
remaining polymer was divided into approximately four equal
portions for reaction with different hydroxylic compounds. The
following table indicates the particular hydroxylic compound which
was used and the amount.
TABLE I ______________________________________ Hydroxyl EDC.sup.3
Compound.sup.1 mg mmoles Solvent.sup.2 ml mg mmoles
______________________________________ HNSA 970 4 DMF 30 786 4 PCP
1064 4 CH.sub.2 Cl.sub.2 20 768 4 OSu 461 4 CH.sub.2 Cl.sub.2 20
768 4 ONp 557 4 CH.sub.2 Cl.sub.2 20 768 4
______________________________________ .sup.1 HNSA --
4hydroxy-3-nitrobenzene sulfonic acid. PCP -- pentachlorophenol OSu
-- N--hydroxy succinimide ON.sub.p -- pnitrophenol .sup.2 DMF --
N,N--dimethyl formamide CH.sub.2 Cl.sub.2 -- dichloromethane .sup.3
EDC -- 1ethyl-3-(3"-dimethylaminopropyl) carbodiimide
hydrochlorid
The four preparations were allowed to react for 24 hours at room
temperature and the resins were then washed with about 30 ml each
5.times.CH.sub.2 Cl.sub.2 ; 5.times.DMF; and 5.times.CH.sub.2
Cl.sub.2. These preparations were dried in in vacuo and then used
directly for cleavage of the oligopeptide from the resin.
Each of the above prepared resins (200 mgs each) were suspended and
stirred in 2 ml anisole cooled to -70.degree. C. (dry ice/acetone)
and purged with nitrogen. An HF lecture bottle valve was opened for
one minute and HF was allowed to condense into the sample. The
cooling bath was then changed to ice water and the temperature
allowed to rise for about two hours to a final temperature of
ambient temperature, while maintaining a nitrogen atmosphere. The
resulting slurry was then extracted with diethyl ether and
suspended directly between layers of 5 ml of borate buffer, pH 8.5,
containing 30 mg of BSA and 10 ml of ether. After decanting the
ether layer, the protein solution was stirred overnight, filtered,
dialyzed extensively for several days against buffer, and
chromatographed on Sephadex G-25 column to remove unreacted
peptide. Excellent yields were obtained with substantial recovery
of any unreacted protein.
The resulting product could be used as an immunogen conjugate for
producing antibodies to the oligopeptide.
In the next example, the peptide FMOC-Lys (.epsilon.-DNP)
-Ala-(P)((P) is the 4-alkoxybenzyl resin) was synthesized and
deprotected by previously described methods for this resin. (See
Chang et al., supra and Meienhoffer et al., supra.) Four separate
portions (500 mg) of the above indicated resin, substituted at a
concentration of 0.133 moles/g were succinylated, washed and
derivatized with the previously indicated hydroxy compounds in
substantially the same manner as described for the chloromethyl
resin. The activated peptide resins were treated with 20 ml of 40%
trifluoroacetic acid in CH.sub.2 Cl.sub.2 for 1 hr. The resin was
filtered off, the solvent removed in vacuo and the residue treated
with 0.1 N borate buffer, pH 8.5, containing 76 mg of BSA. The
resulting product was worked up as described above.
Substantially the same results were obtained with the
4-alkoxybenzyl resin employed in this example as was obtained in
the chloromethyl resin example described previously.
In the next example, a fragment of a viral structural protein
(H-Ile-Pro-Ile-Pro-Ser-Ser-Trp-Ala-Phe-[P]) was synthesized,
deprotected and neutralized on chloromethyl resin as described
previously. One gram of the resin containing 0.3 mmoles of the
peptide was suspended in 15 ml of CH.sub.2 Cl.sub.2, 150 mg of
succinic anhydride added and the mixture stirred overnight at room
temperature. The resulting product was washed with 15 ml each time
5.times.CH.sub.2 Cl.sub.2 ; 5.times.DMF; 5.times.CH.sub.2 Cl.sub.2.
The succinylated resin was suspended in CH.sub.2 Cl.sub.2 (15 ml)
and 256 mg of PCP and 190 mg EDC added. After stirring for 6 hours,
the resin was washed 15 ml each time 5.times.CH.sub.2 Cl.sub.2 and
dried in vacuo.
The activated peptide resin (200 mg) was cleaved using the mild HF
treatment described previously. The mixture of the cleaved resin,
peptide and anasole was suspended between the layers of 5 ml of
0.1N borate buffer, pH 8.2, containing 10 mg of keyhole limpet
hemocyanin, and 5 ml of diethyl ether. The ethereal layer was
decanted and an additional 10 ml of ether added. After stirring the
mixture overnight in the cold (4.degree. C.), the layers were
separated, the aqueous layer dialyzed against phosphate buffered
saline, pH 7.2, and used for immunization. In a repeat of the above
experiment, employing .sup.14 C labeled succinic anhydride, 0.83 mg
of the peptide was found attached to 10 mg of the protein.
In a similar fashion several peptides were synthesized, activated,
cleaved from the polymer and attached to carrier proteins. The
following is a list of such peptides:
a. Cys-Thr-Lys-Pro-Thr-Asp-Gly-Asn-Cys.
b. (a)-Thr-Cys-Ile-Pro-Ile-Pro-Ser-Ser-Trp-Ala-Phe.
c. Gly-Asn Cys-Thr-Cys-Ile-Pro-Ile-Pro-Ser-Ser-Trp-Ala-Phe.
d.
Thr-Lys-Pro-Thr-Asp-Gly-Asn-Cys-Thr-Cys-Ile-Pro-Ile-Pro-Ser-Ser-Trp-Ala-Ph
e.
In the next example, employing chloromethyl resin, substituted with
Boc-Ala-OH(0.32 meq/g), the Ala was sequentially coupled with
Boc-Lys-(.epsilon.-DNA)-OH employing substantially the same
conditions as described above. The mild HF treatment previously
described yielded 0.24 mmoles of succinyl-Lys
(.epsilon.-DNP)-Ala-OH per g (75% yield based on alanine). Four 200
mg portions of the peptide resin containing succinylated peptide
were treated with six-fold excesses of the hydroxy compounds listed
in Table I. After appropriate washes and work up as described
previously, the resin was cleaved by mild HF treatment and after
diethyl ether washes, the peptide-resin mixture was directly
treated with 7 ml of 0.1N borate buffer containing 30 mg BSA and
the pH adjusted to 8.5. The calculated molar ratio of activated
peptide per lysine residue in BSA was 1.8. The protein solution was
filtered, dialyzed extensively, and chromatographed on Sephadex G15
in 0.1N ammonia to remove traces of unreacted peptide. The protein
recoveries were nearly quantitative.
In the next example, 4-alkoxybenzyl alcohol resin was employed and
the same peptide as in the above example was synthesized using FMOC
derivatives, FMOC-Ala as the first attachment, followed by
FMOC-Lys(.epsilon.-DNP)OH and succinic anhydride.
Portions of dried resin were cleaved with TFA/CH.sub.2 Cl.sub.2 and
found to yield under isocratic conditions, 0.133 mmoles peptides
per gram of resin. Four separate portions of 500 mg of resin where
derivatized to form the esters previously described in Table I. The
esters were cleaved with TFA/CH.sub.2 Cl.sub.2, filtered off, the
resin dried and the residue treated with 10 ml of 0.1N borate
buffer, pH 8.5 containing 76 mg BSA. The calculated ratio of
activated peptide to lysine groups in BSA was 1. The reacted
protein was treated as above and results are shown in the following
Table II:
TABLE II ______________________________________ 4-Alkoxybenzyl
alcohol resin.sup..beta., Chloromethyl resin TFA cleavage, mild HF
cleavage, Boc-System.sup..alpha. FMOC-system Efficiency Efficiency
of coupling of coupling Moles DNP (% of Moles DNP (% of Ester
Substituted/ reagent substituted/ reagent used Mole BSA utilized)
Moles BSA utilized) ______________________________________ HNSA 2
2.8 3 4.9 Pcp 16 14.8 12 19.8 OSu 4 3.6 8 13.2 ONp 1 0.9 2 3.3
______________________________________ .sup..alpha. Starting ratio
of peptide to Lys of BSA was 1.8:1. .sup..beta. Starting ratio was
1:1.
It is evident from the above results, that a simple rapid and
efficient method is provided for conjugating polypeptides to a
protein. The method permits substantially quantitative utilization
of the usually scarce and expensive protein reagent, so that
economic conjugations are achieved. Furthermore, the reagents used
are simple and allow for retention of the natural structure and
conformations of the protein conjugate. Also, the particular site
of conjugation of the peptide can usually be selected by employing
appropriate protection and deprotection means known in the art. The
carboxyl group which is used for forming peptide bonds is a
particularly convenient functionality, which can be readily
activated by employing an ester capable of forming peptide bonds in
an aqueous medium. The method does not involve any separation step
from the resin which is employed, until after the conjugation has
been performed, at which time, the resin is easily removed from the
product.
Because of the controlled addition and protection, the method
provides for preparation of concatemers of peptides, as well as
intramolecular cyclization. These compounds can find a variety of
uses in mimicking naturally ocurring compounds, in providing novel
polymers of repeating sequences, and the like.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *